Ultrastructural Changes during the Life Cycle of Mycoplasma salivarium in Oral Biopsies from Patients with Oral Leukoplakia

Abstract

Bacteria in genus Mycoplasma spp. are the smallest and simplest form of freely replicating bacteria, with 16 species known to infect humans. In the mouth, M. salivarium is the most frequently identified species. Mycoplasma spp. are parasites with small genomes. Although most of the Mycoplasma spp. that infect humans remain attached to the host cell surface throughout their life cycle, we have previously reported the presence of Mycoplasma salivarium in the epithelial cells of oral leukoplakia and oral lichen planus. However, the mechanism underlying the pathogenicity of M. salivarium has remained unclear. Further studies are needed to identify the process of infection of human cells and the stages in the life cycle of M. salivarium. Electron microscopy (EM) is the method of choice for morphological investigation of Mycoplasma spp. in cells or tissues. This study was performed to clarify and detail the ultrastructure of M. salivarium in tissue biopsies of oral mucosal leukoplakia, using three EM methods: (1) a standard EM processing method; (2) an ultracryotomy and immunolabeling method; and (3) the LR White resin post-embedding and immunolabeling method. This study included five oral leukoplakia tissue samples showing hyperplasia and hyperkeratosis. Although there was some variation in ultrastructural appearances between the three EM methods used, there were four ultrastructural appearances that are believed to reflect the stages of the M. salivarium life cycle in the epithelial cells of the oral mucosa: (1) small, electron-dense cellular-like structures or elementary bodies of M. salivarium; (2) large structures of M. salivarium; (3) M. salivarium organisms in cell division; (4) the sequence of events in the life cycle of M. salivarium that includes: (a) elementary bodies of M. salivarium deep in the oral mucosal epithelium; (b) replication by binary fission and daughter cell division from the elementary bodies; (c) maturation or degeneration of M. salivarium in the epithelial cells mainly in the upper part of the epithelium; and (d) death of the organisms in the granular and/or keratinized layer. These ultrastructural images may provide a useful reference for the identification of M. salivarium in diagnostic cytology or biopsy material.

Keywords: Mycoplasma; Mycoplasma salivarium; electron microscopy; immunoelectron microscopy; infection; life cycle; oral leukoplakia; oral mucosa.

Figure 1

A flowchart showing the process of sample preparation for electron microscopy in each method.

Figure 2

Light microscopy and immunohistochemistry of Mycoplasma salivarium. (A) A photomicrograph of a hematoxylin–eosin stained tissue section of oral mucosal tissue with oral leukoplakia by light microscopy. The section shows hyperplasia and hyperkeratosis, but there is no dysplasia and Candida is not present (×100). (B–D) Immunohistochemistry of an adjacent section to that shown in (A). (B) Immunohistochemistry shows that the antibody to M. salivarium, which is demonstrated as numerous fine granular stained areas (brown), localizes from the middle part to the upper part of the epithelium, except the keratinized layer (×400). (C) Immunohistochemistry showing that the antibody to M. salivarium localizes at many sites in the granular layer (×1,000). (D) Immunohistochemistry showing the antibody to M. salivarium localizes at fewer sites in the middle part of the epithelium than at the upper part of the epithelium (×1,000). (E) Hematoxylin and eosin staining for light microscopy of a section of normal appearing oral mucosal without hyperkeratosis and epithelial dysplasia (×100). (F) Immunohistochemistry of an adjacent section to that shown in (E) shows no positive immunostaining with the antibody to M. salivarium in the epithelium (×100).

Figure 3

Electron microscopic images of the upper part of the oral mucosal epithelium in samples of oral leukoplakia prepared by the standard electron microscopy method. (A,B) Round or oval Mycoplasma spp. images with various types of internal structures are observed. Arrows indicate several types of Mycoplasma spp. images. Scale bar: 2 μm (A), 1 μm (B), N: nucleus. (C) Large Mycoplasma spp. images shown in (A). The arrowhead indicates a Mycoplasma spp. image containing homogeneous and high electron-dense internal components and the arrow indicates that containing internal components composed of both electron-dense and empty areas. Scale bar: 0.5 μm. (D) Mycoplasma spp. images containing many vacuoles within an image. Scale bar: 0.2 μm. (E) Mycoplasma spp. images having low electron-dense internal components with numerous fine granules (lower) or many clusters of fine granules (upper). Scale bar: 0.2 μm. (F) Round or ovoid structures appearing to be complete vacuoles in the keratinized layer, which are most likely to be the remains of dead Mycoplasma spp. cell. Scale bar: 0.5 μm.

Figure 4

Electron microscopic images of the middle part of the oral mucosal epithelium in samples of oral leukoplakia, which are prepared by the standard electron microscopy method. (A) Many Mycoplasma spp. cells are observed in the cytoplasm of the epithelial cells as round or ovoid electron-dense structures. Scale bar: 4 μm. (B) An image of Mycoplasma spp. cell with high electron density, which is morphologically similar to a large electron-dense image shown in Figure 3C. Its border is clear or poorly defined. Scale bar: 0.2 μm. (C) An image of Mycoplasma spp. cell composed of two parts, one with high electron density and the other with slightly lower electron density, which seems to be in the process of cell division. Scale bar: 0.5 μm. (D) An image of an electron-dense structure coupling with a slightly lower electron-dense structure (daughter cell) (asterisk), which seems to demonstrate cell division of Mycoplasma spp. Scale bar: 0.5 μm. (E) A large, slightly lower electron-dense image including a small area with high electron density in the middle part of the prickle cell layer. This image appears to be a Mycoplasma spp. cell expanding from a small, high electron-dense structure to a large structure with slightly low electron density. Scale bar: 0.5 μm. (F) An image of twin structures, which seem to be on the process of cell division. Scale bar: 0.5 μm.

Figure 5

Electron microscopic images of the oral mucosal epithelium in samples of oral leukoplakia prepared by the ultracryotomy-immunolabeling method. (A,B) Mycoplasma spp. cells are observed as pleomorphic, electron-dense images at low magnification, which vary in size. Scale bar: 2 μm. (C) High magnification image of a Mycoplasma spp. cell with homogeneous and high electron-dense internal components. Scale bar: 0.5 μm. The border of the image is clear or poorly defined. (D) An image of Mycoplasma spp. with internal components containing numerous small lower electron-dense granules. Scale bar: 0.5 μm. (E) An image of Mycoplasma spp. with less electron-dense internal components containing many holes, which vary in number and size. Scale bar: 0.5 μm. (F) Small, round or oval images of Mycoplasma spp. cells with homogeneous and electron-dense internal components in the middle part of the epithelium. Scale bar: 0.2 μm.

Figure 6

Electron microscopic images of Mycoplasma spp. cells, which seem to be in the process of cell division (A–E) and localize in the keratinized layer (F), by the ultracryotomy-immunolabeling method. (A) An image with the appearance of invagination of the membrane, which seems to be in the process of cell division in binary fission. The arrow indicates invagination of the membrane. Scale bar: 0.5 μm. (B) An image of two cells connected by a narrow area, which seems to be on the final stage of cell division. Scale bar: 0.5 μm. (C) An image of a cell dividing in binary fission by invagination, which seems that a daughter cell is dividing from an elementary body. Scale bar: 0.5 μm. The arrow indicates invagination of the membrane. (D) An image showing formation of a septum across the structure, which is recognized to be in the process of binary fission. The arrow indicates a septum across the structure. Scale bar: 0.2 μm. (E) A large and slightly lower electron-dense image including a small area with high electron density in the middle part of the prickle cell layer. This image appears to be a Mycoplasma spp. cell enlarging from a small, high electron-dense structure to a large structure with slightly low electron density. Scale bar: 0.5 μm. (F) An image of round or ovoid vacuole in the keratinized layer, which is not bound by gold particles. Scale bar: 0.2 μm.

Figure 7

Electron microscopic images of Mycoplasma spp. by the LR White resin embedding-immunogold labeling method. These images are very similar to those identified by the standard electron microscopy method. Images of Mycoplasma spp. are bound to gold particles, with a variable amount of the image or part of an image. Many particles are attached on the electron-dense images or areas, while fewer or no particles are attached to the low electron-dense images or areas and vacant areas. Scale bar: 0.5 μm (A,B), 0.2 μm (C–F).

Figure 8

The relationship between the electron microscopic images of the Mycoplasma spp. by the ultracryotomy method and those by the standard electron microscopy method. The differences between images resulting from these techniques are considered to result from the discrepancy of the degree in shrinking and transformation of the micro-organisms, which was caused by fixation, dehydration, and embedding. High electron-dense images (a) are produced from the cells with amorphous and dense internal components (A). Low electron-dense images (b) are produced from the cells with less dense internal components (B). Combination images (c) are assumed to be produced from pleomorphic cells (C). The ratio of the cell surface area and volume of the internal component is larger for the pleomorphic cells (C) than the spherical or oval cells (B). That is, when the volume of the internal component is the same between the cell (B) and the cell (C), the surface area of the cell (C) is larger than the cell (B). Therefore, the discrepancy between the capacity of a cell and the volume of internal components may have arisen by shrinking and transformation from a pleomorphic to a spherical or oval shape. Vacuolated images (d) may be produced from the Mycoplasma spp. (D), which contain many vacuoles within a cell (like a “Swiss cheese”), by shrinking of internal components among the vacuoles.

Figure 9

The proposed life cycle of Mycoplasma salivarium in the epithelial cells of the oral mucosa in oral leukoplakia. The elementary bodies of Mycoplasma salivarium invade into the surface epithelium of the oral mucosa without hyperplasia/hyperkeratosis, and through the epithelial cells and/or via the intercellular spaces, to replicate in the epithelial cells mainly at the middle part of the epithelium. Daughter cells divide from the elementary bodies by binary fission, mature to large cells and degenerate. The maturation and degeneration may proceed in the epithelial cells with migration from the middle part to the upper part of the epithelium, where they die in the granular layer and/or keratinized layer. Maturation of elementary bodies to large cells by expanding is also suggested. This proposed sequence of events in the life cycle of Mycoplasma salivarium in the epithelial cells of the oral mucosa in oral leukoplakia is derived from the observations made in the present study.